UNRELATED DONOR HEMATOPOIETIC TRANSPLANTATION

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Allogeneic hematopoietic stem cell trans-plantation (HSCT) is the treatment of choicefor a range of malignant and non-malignantdiseases. The last decade was marked byprogress in the better definition of the humanleukocyte antigen (HLA) system and theimmunology of transplantation. Improvedclinical results have led to the use of hema-topoietic transplantation at earlier stages ofthe natural history of several diseases and

an extended family search usually identifies a one HLA locus-mismatched relative in less than 5% of cases. The decline in the fertility rate seen in several western countrieswill most certainly aggravate the problem in the future. As a result, most patients in need of an allogeneic marrow transplantrequire an unrelated donor. The number of unrelated HSCT is increasing and accounts for approximately 25% of the allogeneic transplants currently reported tothe International Bone Marrow TransplantRegistry (IBMTR) (1). Here, we review some

UNRELATED DONOR HEMATOPOIETIC

TRANSPLANTATION

Marcos de LimaRichard Champlin

Allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of choice for a range of malignant and non-malignant diseases. Unfortunately, fewer than 30% of patients have a human leukocyte antigen (HLA)-matched sibling. Advances in our understanding of the HLAsystem and the development of large international donor registries are supporting the increasing use of unrelated donors as an alternative source of stem cells. Unrelated donor transplantation,however, is still associated with higher complication rates than in HLA-identical sibling donor transplants. Improvements in graft-vs.-host disease prevention and treatment, new conditioning regimens and better donor selection will likely expand the indications of unrelated donor HSCT inthe next decade.

Advances in the understanding of theHLA system and the development oflarge international donor registries aresupporting the increasing use of unre-lated donors as an alternate source ofstem cells.

the number of patients inneed of HSCT will likelyincrease in the near future.An HLA-matched sibling is available to fewer than30% of patients in NorthAmerica and Europe, and

basic concepts involvedwith hematopoietic trans-plantation from unrelateddonors and summarizerecent results of unrelateddonor transplantation inadults.

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�cell donors have been recruited worldwideand more than 11 000 transplants have beenfacilitated (9).

The Bone Marrow Donors Worldwide(BMDW) database was created in order tofacilitate donor searches internationally,combining HLA data collected from 47 bonemarrow donor registries from 37 countries,and 22 cord blood registries from 15 coun-tries. The BMDW database listed 6 726 454donors and cord blood units as of Novem-ber, 2000, typed for HLA-A and HLA-B, with approximately 40% typed for HLA-DR(10, 11).

HISTOCOMPATIBILITY

Histocompatibility antigens are cell surfacedeterminants that mediate immune reactionsand graft rejection after transplantationbetween genetically diverse individuals.They are widely expressed and are encodedby genes within a chromosomal regiontermed the major histocompatibility complex(MHC), or the HLA complex in humans(17, 18). The HLA complex is located at thep21.3 band of chromosome 6 and containsover 200 genes. HLA genes are concernedwith the immune response. The major loci are grouped into class I and II. The HLA complex also includes class III genes, but their role in transplantation is less established.

Cell surface molecules encoded by HLAgenes mediate cellular interactions during an immune response. Antigen presentingcells process antigens intracellularly, cleav-ing them into peptides which are displayedin the antigen presenting groove on thesurface of an HLA molecule at the cellsurface. T cells will recognize the antigenicpeptide fragments associated with the HLAmolecules. This antigen presentation func-tion of HLA molecules relates to the broadpolymorphism of the MHC: the greater thevariation, the greater the assortment of pep-

HISTORY OF UNRELATEDDONOR TRANSPLANTATIONAND THE DEVELOPMENT OF

DONOR REGISTRIES

The first related transplants were performedin 1968 in two children with severe com-bined immunodeficiency and the Wiskott–Aldrich syndrome. In 1973, Speck et al. (2)reported the first unrelated donor marrowtransplant, in a patient with aplastic anemia.The donor was HLA-A-, HLA-B-matched and mixed lymphocyte culture compatible,but engraftment was not documented. Other attempts at transplanting unrelateddonor marrows were reported after that, but the feasibility of the procedure was conclusively shown in 1979, when a 10-year-old patient with acute lymphoid leukemia(ALL) in second remission received an HLA-A-, HLA-B- and HLA-D-matched unrelated donor transplant. She achieved a complete remission that lasted for 2years, without graft-vs.-host disease (GVHD)(3–6).

The Anthony Nolan Bone Marrow Trustwas the first large registry formed to identifyunrelated donors for hematopoietic trans-plantation. It was established in London in1974, due to the initiative of Shirley Nolan,who began her appeal for volunteer donorsin an attempt to save the life of her son,Anthony, a patient with Wiskott–Aldrich syn-drome (7). Similar programs were started inthe USA in Milwaukee, Saint Paul, Seattleand Iowa City (8). These registries wereestablished by approaching previously HLA-typed platelet donors. In 1984, the USCongress authorized the development of aregistry and, in 1986, the Office of NavalResearch awarded the first contract to estab-lish the National Bone Marrow Donor Registry (NMDP) in Minneapolis, Minnesota.The first NMDP-facilitated transplantoccurred in 1987. Local and national registries were formed in Europe, NorthAmerica, Asia and Australia. Since then, over4 million potential marrow and blood stem

M. de Lima, R. Champlin: Unrelated donor hematopoietic transplantation

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tides that can be presented to lymphocytes(19). HLA molecules have been found to bethe targets for graft rejection and GVHD.

Class I molecules have an a polypeptidechain, encoded by class I genes, and a bchain, encoded by the b2 microglobulingene on chromosome 15. Three ‘classic’class I genes, HLA-A, HLA-B and HLA-C, areof marked importance in the transplantationprocess (20, 21). The World Health Organi-zation (WHO) Nomenclature Committee forFactors of the HLA System reported in 1999the number of presently known HLA class Ialleles: 119 HLA-A, 245 HLA-B and 201 HLA-C (20, 22, 23).

Class II molecules have an a and bpolypeptide chain encoded by class II genes,designated by the class, family and chain,respectively. Individual genes are identifiedby numbers and alleles by numbers pre-ceded by an asterisk (19, 22, 24). Class IIgenes are known as HLA-D region genes,indicating their identification after the dis-covery of HLA-A, HLA-B and HLA-C loci.HLA-D genes code for antigens responsiblefor the activation of T cells in the mixed lymphocyte culture (25). There are five families of class II genes: DR, DQ, DO, DNand DP, comprising nine loci (DRA, DRB1,DRB3, DRB4, DRB5, DQA1, DQB1, DPB1and DPA1) (21). HLA-DR molecules have a non-polymorphic a (A) chain bound to apolymorphic b (B) chain that is encoded by one of four genes (DRB1, DRB3, DRB4and DRB5). HLA-DRB1 and DQB1 are potenttransplant-related antigens, and DRB1 is themost diverse; 201 HLA-DRB1 and 33 HLA-DQB1 alleles have been reported (23).

HLA genes are usually inherited as a unit within chromosome 6; this unit is thehaplotype. The combination of haplotypesinherited from each parent is the genotype.Considering that most humans are heterozy-gous in the HLA loci and that each haplo-type is coexpressed, two diverse antigenswill be coded by a certain locus.

Another peculiarity of the HLA system isthe phenomenon known as linkage disequi-

librium (26). Some groups of alleles are seenin connection with each other more oftenthan would be expected by random associ-ation, a relationship that probably reflectsevolutionary selection. In the presence oflinkage disequilibrium between HLA loci,matching for two of them will frequentlyimply compatibility for a third locus and possibly also for other untested genes. Anexample is the haplotype A1, B8 and DR3,recorded in approximately 5% of Caucasianslisted in the NMDP (27). Conversely, HLA-DP is in very weak linkage equilibrium withHLA-A, HLA-B, HLA-DR and HLA-DQ. Lessthan 20% of HLA-A-, HLA-B-, HLA-DRB1-,HLA-DQB1-matched pairs are matched for HLA-DP (28).

HLA typing

The HLA system was initially described afterthe observation that sera from multiparouswomen or from recipients of multiple trans-fusions induced agglutination reactions ofunrelated donor leukocytes (17, 29, 30). Classically, HLA class I antigens have beenserologically defined on peripheral bloodmononuclear cells (PBMC) by a complement-dependent microcytotoxicity assay usingpanels of alloantisera containing HLA anti-bodies, or by the use of monoclonal anti-bodies. HLA alloantigens so defined mayexpress multiple epitopes. Cross-reactivityoccurs when different antigens share sero-logically specified epitopes (‘public speci-ficities’), forming ‘cross-reactive groups’ or‘CREG’.

Class II antigens have been characterizedby serology for DR and DQ on PBMCenriched for B lymphocytes. Differences inclass II HLA-D antigens may also be evalu-ated by assays, such as the mixed lympho-cyte culture (MLC) (22, 31). The cellularantigens involved in the MLC are combina-tions of HLA-DR, HLA-DQ and HLA-DPdeterminants, with DR being the mostimportant. Considering its low predictive

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typing for HLA-A and HLA-B may becomeroutine in the near future alongside DRB1typing. Currently, molecular HLA-A, HLA-Band HLA-C typing is usually performedwhen the resolution of serologic typing is inadequate.

In the sequence-specific primers (SSP)method (37), a panel of primers is used toamplify a series of known sequences codedby a specific locus and the PCR products arethen submitted to electrophoresis. HLA lowresolution typing may be performed rapidly,but high resolution typing requires a largenumber of PCR considering the polymor-phisms involved. Sequence-specific oligonu-cleotide probe (SSOP) hybridization assaysuse oligonucleotide probes labeled withradioactive or non-radioactive molecules thatare hybridized with the PCR-amplified HLAgene to be evaluated. Only probes that arecomplementary to the DNA will hybridizeand the resulting pattern will determine theHLA type. Sequencing methods provide theutmost level of resolution. Alleles and genesmay be studied from cloned templates orfrom PCR-amplified genomic DNA (22, 35,36). Fluorescence automated sequencingtechnology uses allele-specific or locusprimers to amplify the HLA allele or gene of interest from genomic DNA. The templateDNA is sequenced by PCR and the strandsare detected by linking them to fluorescentlabels. After electrophoresis, an automatedDNA sequencer reads the fragments.

REGISTRIES AND THE SEARCH PROCESS

The process of identifying a compatibledonor is termed the ‘unrelated donor search’.A preliminary computer consult initiates thesearch process. The patient’s HLA typing iscompared to those listed in the registry andpotential donors are then identified who areHLA-A-, HLA-B- and DRB1-matched, andalso those who are HLA-A- and HLA-B-

value for both GVHD and rejection, theNMDP does not require an MLC in matchedunrelated donor transplants. Serologicmethods of HLA typing have several limita-tions. Lack of specificity of antisera is a majorintrinsic limitation that may improve with theuse of monoclonal antibodies. Lack of anti-sera recognizing some antigens expressed incells of non-Caucasians is another problem.Because viable B lymphocytes are necessaryfor class II serologic typing, lymphocyto-penia may preclude class II typing. Molecu-lar techniques have evolved rapidly and, in the light of the limitations of serologic and cellular assays, are replacing the olderapproaches to HLA characterization. Molec-ular typing has led to the identification ofnumerous class I and II alleles that could notbe defined by serologic typing. The numberof serologic equivalents is 90, 190 and 145,respectively, for HLA-A, HLA-B and HLA-DRB1. These numbers correspond to 76%,77% and 72% of the known alleles for these loci (23).

A donor–recipient pair matching for thetwo alleles at HLA-A, HLA-B and HLA-DRB1is a ‘six-antigen match’. Matching for HLA-Cand HLA-DQB1 is frequently considered,especially in the context of unrelated donortransplantation, and is a ‘10-antigen match’.

HLA typing based on serology does notdetect the polymorphism determined bymolecular methods which define class I and II alleles. DNA typing characterizes thegenotype coding of the unique sequences in a molecule. Polymerase chain reaction(PCR)-based methods may directly establisha coding region sequence of an allele or may infer it by exclusion or confirmation ofspecific sequence polymorphisms (32–34).Low resolution DNA typing has the samelevel of HLA characterization as serologictyping. High resolution DNA typing or mol-ecular typing denotes allele level identifica-tion. Class I molecular typing is a moredifficult task than class II typing, due to thehigher polymorphism and the presence of pseudogenes (35, 36), but molecular


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matched in whom DRB1 typing has not beenperformed. The proportion of donors whoseHLA-A, HLA-B and HLA-DR are knownvaries in different registries. As of December2000, the NMDP register included 3 196 174donors; 100% HLA-A and HLA-B typed and66% HLA-A, HLA-B and HLA-DR typed (9).To continue the search process, an NMDP-approved transplant center must request aformal search in which further DR typing isperformed if necessary to identify an HLA-A, HLA-B and HLA-DRB1 compatible donor,and a second blood sample from potentiallymatched donors is obtained for confirmatorytyping at the transplant center. Recently,NMDP has instituted a requirement for mol-ecular class I typing, as well as oligonu-cleotide typing for HLA-DRB1 alleles. Recentdata indicate that optimal results occur withHLA class I and II molecularly matcheddonor–recipient pairs. Unfortunately, a perfectly matched donor is not available for most patients and registries allow for theselection of unrelated donors with varyingdegrees of mismatching.

Investigational Study and an InvestigationalNew Drug Application accepted by the Food and Drug Administration (FDA),making all registered donors eligible forgranulocyte colony stimulating factor (G-CSF) mobilization of PBSC. The patient’stransplant team makes the decision aboutwhich source of stem cells to use. Thepatient is responsible for all the costsinvolved in HLA typing, donor evaluation,etc. At the MD Anderson Cancer Center, thetypical cost for a donor search ranges from$6000 to $10 000 depending on the numbersof donors undergoing DR or confirmatorytyping.

HLA alleles are not randomly distributedbecause of linkage disequilibrium. The fre-quency of HLA alleles and linked haplotypesvaries among racial groups. Approximately10% of patients have very common haplo-types; unrelated donors can readily be foundfor such individuals. Unfortunately, approx-imately half of patients have uncommonhaplotypes and most will not have a donoravailable in registries worldwide. In general,the chance of finding a donor is best amongindividuals of the same genetic background.Identifying a donor is a major challenge forracial minorities and ethnic groups under-represented in the donor registries.

The search process and preparation of thedonor for transplant generally requires2–6 months. Patients with advanced hema-tologic malignancies are inherently unstable.New medical complications or disease pro-gression may occur in the recipient duringthe time required for the search process.Funding for typing, search and marrowacquisition may not be available or beobtained only after significant delays. Evenwhen a potential donor is identified, up to5% of donors may have a medical conditionpreventing donation and some may beunavailable or unwilling to donate (13, 14).The median time from initiating a search to performing an urgent transplant is4–6 weeks if a class I and II compatibledonor is identified in the initial computer

Unrelated donor hematopoietic stem cells canpotentially extend the benefit of transplantationto all patients in need.

If confirmatory typing is acceptable, theprospective donors are ‘activated’ and prepared for the transplant. They are inter-viewed, medically evaluated and checkedfor transmissible infectious diseases. Whenthe donor has clinical clearance from the NMDP-approved donor or collection center, the information is passed to thepatient’s transplant team. The marrow dona-tion harvest surgery is performed and thegraft is shipped to the recipient’s hospital(9, 10, 13, 14). Overall, 75% of NMDP donorsreceive general anesthesia, 18% receiveepidural anesthesia and 7% receive spinalanesthesia. On average, 1 L of marrow isremoved, but the exact amount is deter-mined by the recipient’s weight, taking intoaccount the donor’s size. The NMDP has an

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search, and up to 6 months if additionalHLA-DR typing of potential donors isrequired.

The success in finding DRB1-matcheddonors is directly related to the allele prevalence in the population, and the combination of class I and II alleles in-herited by the subject. The probability of identifying a DRB1-matched donor amongserologically matched HLA-A, HLA-B, HLA-DR donors depends on the number ofdonors so identified. It ranges from 40% ifonly one serologically matched donor isfound, to 90% if five donors are available(12).

Specific requirements for the donation ofunrelated marrow or peripheral blood stemcells (PBSC) may vary in each registry, but all donations must be voluntary. TheNMDP limits donations to persons aged18–55 years, in good general health. Alldonors provide informed consent to par-ticipate and are screened for medical contraindications and to preclude the trans-mission of blood-borne infectious diseases.Human immunodeficiency virus (HIV) infec-tion, hepatitis and other active infections are absolute contraindications to donatinghematopoietic stem cells. NMDP and otherregistries initially provided bone marrow as the source of hematopoietic cells for transplantation, but more recently many registries have begun to provide PBSC andsome registries also facilitate cord bloodtransplantation.

PROBABILITY OF FINDING ANHLA-MATCHED DONOR

The impact of racial genetic polymorphismon the chances of identification of an HLA-matched unrelated donor was evaluated byBeatty et al. (15). The authors used theNMDP database that contained, at the timeof the study, HLA types of 20449 patientsand 1625159 potential donors. Haplotype

frequencies were estimated for African–Americans, Caucasians, Asian–Americans,Hispanics and Native Americans (self-reported racial designations) and the pro-bability of finding matched donors wasdetermined according to different hypothet-ical registry sizes. The likelihood of identi-fying a matched donor was 78%, 68%, 60%,62% and 35%, respectively, for Caucasians,Native Americans, Asian–Americans, Hispan-ics and African–Americans. Analyzing thenumber of new HLA phenotypes acquired as a function of new donors incorporated inthe registry, it was documented that newlyrecruited African–Americans are more likelyto express unique phenotypes, a measure-ment of genetic polymorphism. Withinhypothetical registries of each race, 54% ofHispanics and 18% of African–Americanswere projected to find a donor among a 500 000 Caucasians registry. The number ofhaplotypes is greater among African–Americans, while Caucasians and Asian–Americans have a higher frequency ofcommon haplotypes. In Japan, the numberof donors expected to provide an effectiveunrelated marrow donor pool is relativelysmall, illustrating the greater genetic homo-geneity of that population (16). For a 75%chance of identifying an HLA-A-, HLA-B-,HLA-DR-matched donor, 400 000 Caucasiandonors need to be recruited, while forAfrican–Americans this number is 3 million.Therefore, recruiting more minority donorswill improve the odds of finding a donor,but will most likely not result in an equalproportion of donors being found for allraces. Because the definitions of ‘match’ arelikely to change with improvements in tech-nology and are possibly different for differ-ent races, another goal to be pursued is theuse of mismatched donors. Beatty et al. (15)also estimated that, if a one locus mismatchis allowed, registry sizes for a 90% proba-bility of finding a suitable donor are asfollows: 30 000 Caucasians, 30 000 Asian–Americans and 100 000 African–Americans.Cooperation with international registries and

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allele level was detected in 7%, 27% and 39%of 100 potential donor–recipient pairs forHLA-A, HLA-B and HLA-C, respectively.Serologically detected mismatches occurredin 3% and 9% for HLA-A and HLA-B. Thelimitations of serologic resolution at HLA-Bwere clear from the increase in the percent-age of mismatches with molecular typing.HLA-C and HLA-B are in strong linkage dis-equilibrium, and 22 of 25 pairs mismatchedat HLA-B also had disparity at HLA-C (39).

In Caucasian patients and their serologi-cally matched unrelated donors, an analo-gous study revealed that 48% of the pairswere mismatched for one or more alleles atthe HLA-A, HLA-B, HLA-C, HLA-DRB1 andHLA-DQB1 loci. Single mismatches weredetected in 26% of cases, two mismatches in 12% and 10% had disparities for three or more alleles (44, 45).

Engraftment

Primary graft failure occurs when donor-derived hematopoiesis cannot be docu-mented in the recipient after the transplant.Secondary graft failure is defined as a lossof donor-derived hematopoiesis after initialengraftment. Different definitions have beenused, but the major diagnostic criterioninvolves failure to recover or maintain> 0.5 ¥ 109 neutrophils/L. Graft failure isusually due to rejection, but infections ormyelosuppressive drugs may also be thecause. The reported incidence of graft failurefor matched unrelated donor bone marrowtransplant (BMT) is in the range 3–11%(46–48).

HLA incompatibility is a major deter-minant of graft rejection, mediated by im-munologic mechanisms involving residualhost-derived cells (49, 50). Anasetti et al. (49)reported that graft failure occurred in 12.3%of recipients of an HLA-haploidenticalmarrow from family members and in 2% ofpatients who received an HLA-identicalmarrow. The rate was 9% for patients who


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the increased integration through the BMDWregistry are expected to improve the odds ofunrelated donor searches as well (10). As ofOctober 31, 2000, the NMDP had facilitated11422 unrelated HSCT, 1696 of which hadbeen for minority patients.

EFFECT OF HLA MATCHING ONOUTCOME OF UNRELATED

DONOR TRANSPLANTS

Prevalence of class I and II mismatches

Several investigators have found evidencethat allelic disparities frequently occuramong serologically matched donor–recipient pairs, but their impact on HSCT isonly now being fully evaluated with theincreasing use of class I and II allele leveltyping (38, 39). Instances of graft rejectionand GVHD induced by alloresponses due to a single amino acid difference in HLA-B44 have been reported (40, 41), andincreased frequencies of cytotoxic T-lymphocyte precursors occur in HLA-A andHLA-B serologically matched pairs (42).

The prevalence of HLA disparities among440 HLA-A, HLA-B and HLA-DR serologicallymatched Japanese donor–recipient pairs hasbeen retrospectively evaluated by molecularmethods. At least one allele mismatch wasdetected in 26%, 16%, 31%, 18% and 20% ofthe pairs at loci HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1, respectively (43).Overall, 45% of the phenotypically matchedpairs had disparities at one or more HLA-A,HLA-B or HLA-DRB1 alleles.

HLA-C has an important role in modulat-ing natural killer (NK) cell activity. Expres-sion of self-MHC protects cells from NKcell-mediated cell destruction; this interac-tion occurs between one of the domains ofHLA-C and a killer inhibitory receptor on NKcells. Scott et al. (39) incompatibility at the

HLA-DQB1. Recipients of fully matchedmarrows failed engraftment in 2% of cases;a similar incidence was seen among reci-pients of marrows mismatched at a singleclass I HLA-A, HLA-B or HLA-C allele. Nograft failure occurred among patients with asingle class II locus disparity. The presenceof two or more class I allele mismatchesraised the graft failure rate to 29%, while nograft failure occurred among seven patientswith two class II mismatches. Twelve percent of the patients with mismatches in classI and II loci failed to engraft.

Acute graft-vs.-host disease

GVHD-related non-relapse mortality remainsthe primary cause of treatment failure aftertransplantation of T-repleted marrow graftsfrom matched unrelated donors. Similar tothat which takes place in related donor transplants, HLA mismatching increasesGVHD occurrence and severity after unre-lated donor transplants. Petersdorf et al. (12)studied 364 patients who received an HLA-A, HLA-B and HLA-DR serologically matchedunrelated donor transplant, to evaluate theimpact of molecular typing of DRB1 alleleson transplant outcomes. Cyclosporine andmethotrexate were used for GVHD prophy-laxis. The probability of severe acute GVHDwas 48% for the group of 305 molecularlymatched patients and 70% for the 59 DRB1-mismatched subjects. Furthermore, matchingfor DRB1 decreased the overall mortality andthe risk of a transplant-related death. Theauthors pointed out that undetected dispar-ities for class I genes and class II loci DRB3,DRB5 and DQB1 are probably higher amongDRB1-mismatched donor–recipient pairs andcould be involved in the increased GVHDand mortality rates. Accordingly, anotherstudy from the Seattle group clearly definedthe importance of HLA-DQB1 as a trans-plantation antigen (52). The studied pop-ulation consisted of 449 patients whounderwent HLA-A, HLA-B, HLA-DR serolo-


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had one serologically defined incompatiblelocus and 21% for those who had twoincompatible loci. A retrospective study ofpatients with hematologic malignanciescompared rates of graft rejection in recipi-ents of HLA-identical sibling donor, fullymatched unrelated donor and mismatchedunrelated donor transplants. The incidenceswere 5%, 6% and 15%, respectively (47). Mismatches at HLA-DRB1 and HLA-C havebeen shown to correlate with graft failureincidence, as well as HLA-A and HLA-Ballele mismatches (46, 51).

A case control study evaluated the impactof HLA mismatching on engraftment afterunrelated donor transplantation. Twenty-onecases that failed to engraft (86% with chronicmyeloid leukemia (CML)) were matchedwith 42 control patients selected on the basisof variables known to correlate with engraft-ment. HLA-C allele mismatch alone, or incombination with HLA-A or HLA-B mis-match, was documented in 71% of cases and in 33% of the control pairs. Multivariateanalysis gave an odds ratio for graft failureof 4.0 for HLA-C allele-mismatched patients.There was a trend in the direction of anincreased odds ratio with HLA-A and HLA-Bmismatches (51). Graft failure in this settingis probably mediated by recipient class Ireactive cytotoxic T lymphocytes or NK cells reacting because of class I disparities(22, 45). The risk of graft failure is higher in patients with a positive lymphocyte cross-match that detects antibodies againstdonor lymphocytes. Graft failure occurred in62% of 21 recipients of a haploidenticalrelated donor marrow with a positive anti-donor cross-match (against donor T and/orB cells) (45).

The proportion in which class I and II disparities contribute to graft failure wasevaluated in a series of 300 unrelated donortransplants performed for the treatment ofCML (44). HLA matching was performed at the allele level of definition for class IHLA-A, HLA-B and HLA-C, HLA-DRB1 and

logically matched HLA class I and II. DRB1allele typing was performed in the majorityof cases. Retrospective molecular typing ofHLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1 was performed. DPA1 compatibilitywas detected in 76% of donor–recipient pairswith no apparent beneficial effect of match-ing on patient survival or GVHD incidence.DPB1 compatibility occurred in 27% of pairsand significantly influenced survival and theincidence of mild and severe acute GVHD.The assessment of the role of HLA-DP as atransplantation antigen will require a largepopulation in order to interpret the con-founding effects of mismatches at other HLAloci. In fact, the proportional importance ofmatching for different loci in determiningunrelated donor transplant results is yet tobe fully understood. It is also possible thatsome mismatches are more ‘permissive’ thanothers, by virtue of the amino acid changesinvolved or their location in the HLA mole-cule. This would have an obvious impact on donor selection and availability. Table 1summarizes the results of HLA matching and transplantation outcomes.

Chronic graft-vs.-host disease

Chronic GVHD is the most common latecause of morbidity and mortality after allo-geneic HSCT. The prevalence and incidenceof this complication are probably increasing,due to the changes that are occurring in thepractice of blood and marrow transplanta-tion. The use of PBSC has been associatedwith an increased incidence of chronicGVHD in many studies of matched siblingtransplants, and this is a potential concernwith unrelated transplants where there is arelatively high incidence of both acute andchronic GVHD. Donor lymphocyte infusionsmay induce remission in a significant frac-tion of patients with CML relapsing postal-logeneic related and unrelated transplants,but it is associated with chronic GVHD (54).One of the major recent advances in the


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gically matched unrelated transplants andwere retrospectively investigated by SSOPhybridization for HLA-DRB1 and HLA-DQB1.Mismatches were detected in 25% of therecipients; 9% were HLA-DRB1 onlymatched, 11% HLA-DQB1 only mismatchedand 5% were mismatched at both class IIantigens. The probabilities of severe acuteGVHD were 0.61, 0.55 and 0.71, respec-tively, and 0.42 for the 75% of patientswithout mismatches.

The effect of class I allelic mismatches on clinical outcome was investigated bySasazuki et al. (43). They performed retro-spective allele DNA typing of HLA genes in440 Japanese patients who received sero-logically matched HLA-A, HLA-B and HLA-DR unrelated marrow transplants, 80% ofwhom had leukemia (43). Incompatibility forHLA-A and HLA-C alleles was shown bymultivariate analysis to be an independentrisk factor for severe acute GVHD, while thepresence of class II mismatches was not anindependent predictor of GVHD. However,the incidence of grades III and IV acuteGVHD in this study was only 18%, possiblyreflecting a greater genetic homogeneity inthe studied population than in those patientsreported by Petersdorf et al. (12, 52).Sasazuki et al. (43) hypothesized that theimportance of different HLA loci in BMT mayvary according to ethnicity. Linkage disequi-librium may offer another interpretation,however. Because HLA class I allele typingwas not performed in the studies that iden-tified the importance of DRB1 and DQB1 inhematopoietic transplantation, the associa-tion between an elevated risk of GVHD andallelic level class II mismatches could actually reflect class I disparities (43).

The linkage disequilibrium between HLA-DP and other HLA loci is not strong, andmismatches are very frequent among class Iand II serologically or molecularly matchedpairs. The influence of donor–recipientDPA1 and DPB1 matching on transplant out-comes was examined in 122 unrelated BMTpairs (53). Patients and donors were sero-


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field, the use of HSCT in elderly persons, isoften complicated by chronic GVHD, as theincidence rises with increasing recipient age(55, 56). Unfortunately, recent progress inacute GVHD prevention and treatment hasnot translated into significantly reducedchronic GVHD rates.

Chronic GVHD occurs in 30–75% of reci-pients of unrelated donor HSCT (43, 46, 57,58). Prior acute GVHD is a major risk factorfor the development of the chronic form, butunrelated BMT recipients may develop ‘denovo’ chronic GVHD, without acute GVHD.The type of GVHD prophylaxis may influ-ence the risk of extensive chronic GVHD(59–63). The NMDP experience with a largecohort of patients with CML showed an

overall incidence of chronic GVHD of 73%at 2 years, while extensive chronic GVHDoccurred in 60% of the evaluable cases.Lower risks were seen for patients treated inthe chronic phase and with T-cell depletion.Prior grade III or IV acute GVHD was inde-pendently associated with an increased riskof chronic GVHD, with a relative risk of5.9463. Chronic GVHD was reported in55 ± 7% of 462 patients with several diseases,whose transplants were facilitated by theNMDP (64). In 35 ± 7% of cases, it wasextensive.

A longer duration of prophylacticcyclosporine has been suggested to beimportant in avoiding chronic GVHD byallowing time for the development of

Grade III–IV acute GVHD Probability of relapse* Probability of survival*

Reference HLA locus Matched X Mismatched Matched X Mismatched Matched X Mismatched

Petersdorf DRB1 48% X 70% 39%** X 28%** NS 48% X 35%et al. (12)

Sasazuki -A 15% X 31% P < 0.001 20% X 20% P = 0.43 65% X 40% P < 0.001et al. (43)† -B 17% 31% P = 0.006 20% X 20% P = 0.72 60% X 45% P = 0.039

-C 13% 32% P < 0.001 20% X 10% P = 0.06 50% X 50%-DRB1 17% 27% P = 0.058 60% X 60% P = 0.44-DQB1 18% 22% P = 0.46 65% X 50% P = 0.08

MortalityHansen -DRB1 35% X 50% RR = 1 X RR = 2.7et al. (46) P = 0.002

Petersdorf -DQB1 42% 61%et al. (52)†† -DRB1 42% 55% NA NA

-DRB1and DQB1 42% 71%

Przepiorka -DQB1 or 100-day TRMet al. (78)§ -DRB1 22% X 43% 21% X 50%

-DRB1and -DQB1 22% X 64% 21% X 42% P = 0.02

Table 1HLA mismatch and transplant outcome

GVHD: graft-vs.-host disease; HLA: human leukocyte antigen; RR: relative risk; TRM: treatment-related mortality.Recipient–donor pairs serologically matched for HLA-A, -B and -DR. Molecular typing performed retrospectively.* At 1 year.** At 2 years.† Includes one or two mismatches in each loci (majority with one allele mismatched).†† Conditional probability of GVHD.†† Relative risk of grade III–IV aGVHD by HLA mismatch: DQB1 only, 1.8 (95% CI, 1.1, 2.8); DRB1 only, 1.4 (95% CI, 0.92, 2.3); DRB1 and DQB1, 1.9 (95% CI, 1, 3.2).§ GVHD prophylaxis with tacrolimus and mini-methotrexate.

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mortality in the presence of DRB1 mismatchwas 0.51 at 1 year vs. 0.39 for recipients ofmatched marrows, while the probability ofsurvival at 1 year was 0.35 vs. 0.48, respec-tively. Factors that influenced negatively theoverall and transplant-related mortality wereDRB1 mismatch, disease stage (high risk,CML in blast crisis, acute leukemia in relapseand lymphoma; low risk, CML in chronicphase) and age greater than 20 years. No difference in relapse rate was detectedbetween recipients of HLA-DRB1-matchedand -mismatched marrows, implying theabsence of the graft-vs.-leukemia (GVL)effect associated with HLA mismatch, con-trary to suggestions in other studies.However, the relatively small number ofpatients and the heterogeneity of the dis-eases treated complicated the interpretationof the results.

HLA-A allele mismatching was demon-strated to be an independent risk factor fordeath in the study of Sasazuki et al. (43). Thesurvival rate was not influenced by HLA-Cmismatches, despite the increased risk forsevere GVHD. The authors found in the mul-tivariate analysis that matching for HLA-Cwas associated with an odds ratio for diseaserelapse of 2.22. No data were given on HLA-C disparities and graft rejection. Mismatchesfor HLA-DRB1 and HLA-DQB1 class II allelesinfluenced survival only among patients with concomitant class I mismatches. Inter-estingly, isolated disparities at HLA-DPA1and HLA-DPB1 did not impact on survival.

STEM CELL SOURCES

Bone marrow

The vast majority of unrelated donor trans-plants use bone marrow as the source ofstem cells. The results discussed in differentpapers in this volume were generallyobtained with BMT. Hematologic recoveryafter unrelated BMT is well represented by

donor–host tolerance. This approach has tobe weighed against the disease relapse riskthat may increase with prolonged immuno-suppression. The development of extensivechronic GVHD often requires prolongedimmunosuppression with steroids and othermedications. It is an expensive and time-consuming treatment that may preventreturn to work and may seriously compro-mise the patient’s self-esteem and physicalperformance (65).

Non-HLA factors related toincreased incidence of GVHD

Donor–recipient disparity of the minor his-tocompatibility antigen HA-1 has beenreported as relevant for the development ofGVHD in HLA-A*0201-positive subjects (66).The explanation for the development ofGVHD in genotypically matched siblingsmay be due to a mismatch for minor his-tocompatibility antigens, coded by genesoutside the MHC (67). A retrospective studydemonstrated a link between mismatch for minor histocompatibility antigens HA-1,HA-2, HA-4 and HA-5 and the occurrence of GVHD in adult recipients of marrow fromHLA genotypically identical donors. Theseantigens are recognized by T cells in associ-ation with the major histocompatibility anti-gens HLA-A1 and HLA-A2 (68). Their relativecontribution to graft failure and GVHD in thecontext of unrelated donor transplantation isunknown, but it is likely to be significant.

The use of a female donor for a malerecipient increases the risk for grade III orIV GVHD and extensive chronic GVHD. Oneof the possible explanations for this is minorhistocompatibility antigen mismatch. Donorparity increases the likelihood of GVHD and older donor age increases the chancesof both acute and chronic GVHD (62).

Disease relapse and survival

In the series reported by Petersdorf et al.(12), the probability of transplant-related


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the following studies. The Seattle experiencewith 196 patients with CML in chronic phasewho received unrelated BMT has beenreported (46). Ten of 192 available patientshad graft failure. The median time to neu-trophil recovery was 22 days (range,14–51 days); it occurred earlier among sub-jects receiving larger cell numbers (relativerisk of 1.5 for each increment of 1 ¥ 108

cells/kg). The median time to platelet recov-ery was 22 days (range, 5–170 days). HLA-DRB1-mismatched patients showed a longertime to platelet recovery than recipients offully matched transplants, and those receiv-ing larger amounts of donor marrow nucle-ated cells achieved platelet transfusionindependence more rapidly. An analysisfrom the NMDP on 1423 patients with CMLtreated with unrelated donor transplantationfound a primary engraftment failure rate of9.9%, with an additional 6.6% of patientsshowing late graft failure (63). The mediantime to neutrophil engraftment was 20 days(range, 8–42 days).

Peripheral blood stem cells

The number of allogeneic HLA-identicalsibling donor PBSC transplants is increasingrapidly and more than 20% of the allogeneictransplants reported to the IBMTR in 1998used PBSC (1). The safety of G-CSF mobi-lization and collection of peripheral bloodcells from normal donors has been exten-sively reviewed (69). Apheresis productsusually contain 1 log more lymphocytes thando bone marrow harvests. The incidence ofacute GVHD appears to be similar to thatobserved with BMT, but the incidence ofchronic GVHD may be increased. A large retrospective series of patients reported tothe IBMTR and to the European Blood andMarrow Transplant (EBMT) Registry com-pared results of PBSC vs. BMT in the contextof HLA-identical sibling transplants (70).Faster hematologic recovery and shorterinitial admission to the hospital wereobserved with PBSC. The incidence of acute

GVHD did not differ, but the incidence ofchronic GVHD was significantly higher withPBSC. Patients with advanced stage CML orpoor prognosis acute leukemia had a betterdisease-free survival and transplant-relatedmortality with PBSC than with bone marrow,but this benefit was not evident for patientswith less advanced leukemias.

Ethical issues related to the use of G-CSFby donors have delayed the widespread useof PBSC for unrelated transplants. As moredata are accumulated on the safety of stemcell mobilization and collection, the propor-tion of unrelated donor transplants per-formed using PBSC will likely increase in thenear future. The correlation of transplantedcell dose with improved outcomes and thepossibility of obtaining large amounts ofprogenitor cells with apheresis are two otherfactors that suggest that the use of PBSC mayfurther advance the field of unrelated donortransplants.

More than 100 NMDP donors havedonated PBSC to patients who needed asecond transplant. In Europe, more than 150unrelated donors have provided PBSC forprimary unrelated donor transplants (9). Areport on the European experience com-pared outcomes of unrelated donor trans-plants with non-manipulated PBSC (45patients), marrow grafts (45 patients) andCD34-selected PBSC (18 patients) (71). Ashorter time to a platelet count greater than50 ¥ 109/L and to an absolute neutrophilcount greater than 0.5 ¥ 109/L was associ-ated with the use of PBSC. Severe acuteGVHD occurred in 20%, 30% and 18% ofcases, and the probability of chronic GVHDwas 85%, 59% and 0%, in patients receivingmarrow grafts, unmanipulated and CD34-selected PBSC, respectively (P = not signifi-cant (NS) for acute GVHD and P < 0.01 forchronic GVHD). Transplanted-related mor-tality, survival and 2-year relapse-free sur-vival were similar. Despite the low numberof treated patients, unrelated PBSC trans-plant appears to be safe and results in fasterengraftment than with bone marrow. The


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determination of the likelihood of acute and chronic GVHD, as well as the assess-ment of the efficacy of the graft-vs.-tumoreffect with unrelated PBSC, await the treat-ment of a larger number of patients withlonger follow-up.

In addition to potential advantages for therecipients, the donation of PBSC mayprovide additional advantages for the donor,including the avoidance of anesthesia andfaster recovery without multiple bonemarrow aspirations. The theoretical risks ofG-CSF mobilization will only be addressedby an increasing number of donors beingfollowed for a prolonged period of time, in order to detect a possible increased inci-dence of leukemia and other complications(69).

Cord blood

Unrelated cord blood (UCB) contains ade-quate amounts of hematopoietic stem cellsfor stable engraftment in children. In pedi-atric patients, there is a decreased incidenceof GVHD, and transplant results are compa-rable to those obtained with unrelated donormarrow transplants, allowing for greater HLAdisparity (72, 73). Results in adults are lessimpressive, with problems related to delayedengraftment and defective immune reconsti-tution. Graft nucleated cell count is a majordeterminant of survival.

Laughlin et al. (74) have reported theexperience accumulated at five institutionsin the USA with 68 adult subjects trans-planted with UCB. The authors used prefer-ably UCB units matched for at least three ofthe six HLA loci, with a minimum nucleatedcell dose of 1 ¥ 107/kg recipient weight.Seventy-one per cent of patients receivedgrafts disparate at two or more HLA loci.Fifty-four of 60 available patients survivingbeyond day 28 had neutrophil engraftmentat a median of 27 days (range, 13–59 days).Higher UCB cryopreserved total nucleatedcell dose and infused colony forming units

were associated with faster neutrophil recov-ery, and higher infused CD34+ cell dose wasassociated with increased event-free sur-vival. The probabilities of acute GVHDgrades II–IV and III–IV and chronic GVHDwere 0.60 (confidence interval (CI): 0.49,0.71), 0.20 (CI: 0.11, 0.29) and 0.38 (CI: 0.18,0.35), respectively. With a median follow-up of 22 months, the probability of event-free survival was 0.26 at 46 months post-transplant, and the probability of relapse was0.14 (CI: 0.27, 0.01).

The European experience with adult cordblood transplantation has been reported byRocha et al. (75) in abstract form. They ana-lyzed 108 patients reported to Eurocord,with a median age of 26 years and a medianweight of 60 kg. The majority of the recipi-ents had leukemia. All but six patientsreceived mismatched UCB, containing amedian number of nucleated cells of1.7 ¥ 107/kg. By day 60 post-transplant, neutrophil recovery was documented in 81%of patients, and platelet recovery in 65% atday 180. The median times to an absoluteneutrophil count greater than 500/mm3 andto platelets greater than 20 000/mm3 were32 days (range, 13–57 days) and 129 days(range, 26–176 days), respectively. Severeacute GVHD occurred in 27 patients and nocorrelation between GVHD incidence andnumber of HLA disparities was observed, buta significant fraction of recipient–donor pairswere not molecularly typed for HLA-DRB1.Chronic GVHD was observed in 15 of 58patients at risk. Actuarial transplant-relatedmortality at day 100 was 54%. Factors influencing transplant-related mortality bymultivariate analysis were disease status attransplant and nucleated cell content of thegraft greater than 1.7 ¥ 107/kg recipient. Themajor causes of death were lack of engraft-ment and infections (41% of deaths); the 1-year probability of survival was 33% forpatients who received more than 2 ¥ 107

nucleated cells/kg and 39% for patients withless advanced disease at transplant.

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three antigen mismatches. The preparativeregimen consists of fludarabine, rabbit ATGand TBI. Our major goal is to extend the useof cord blood transplantation to adults andlarger individuals who lack more suitabledonors.

GVHD PREVENTION REGIMENS

Cyclosporine, tacrolimus and methotrexate

The combination of cyclosporine, from day–1, and methotrexate, 15 mg/m2 on day 1and 10 mg/m2 on days 3, 6 and 11 post-BMT,is the most commonly used regimen for theprevention of GVHD after matched unre-lated donor transplants. Grade II–IV acuteGVHD occurs in 60–85% of cases and severeacute GVHD in 30–55%. Molecularlymatched donor–recipient pairs have lowerincidences of GVHD than serologicallymatched pairs, as discussed above (12,43–45, 52). The addition of steroids to thecyclosporine and methotrexate regimen hasbeen attempted, but has not improvedresults consistently (59); there are concernsregarding corticosteroid toxicities and pos-sibly an increased risk of infections.

Tacrolimus is a macrolide lactone that is a more potent immunosuppressive agentthan cyclosporine, with similar biologiceffects. The combination of tacrolimus andmethotrexate was compared to cyclosporineand methotrexate in a randomized study andshown to result in a lower rate of acuteGVHD, but with no change in survival (60).A reduced dose of methotrexate, 5 mg/m2

on days 1, 3, 6 and 11 (‘mini’-methotrexate),is commonly employed with similar rates of GVHD. This regimen produces lessmucositis and probably faster hematologicrecovery, and it is the standard regimen for GVHD prophylaxis at our institution.However, it has been relatively ineffective in preventing the development of acute


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The major limitation to the use of cordblood transplants for adults is the lowabsolute number of progenitor cells in thegraft that adversely affects hematopoieticrecovery and survival. A potential advantageis the seemingly decreased incidence ofGVHD, allowing the use of units with greaterHLA disparity, and thereby extending thepossibility of carrying out an HSCT in a large number of patients without related orunrelated marrow donors. Delayed engraft-ment and pancytopenia-related complica-tions are the major causes of death after UCBtransplants, and interventions designed toincrease the number of UCB cells are likelyto improve the results. Ex vivo expansion isunder investigation and another approachhas recently been reported by Barker et al.(76) in abstract form. They documented adouble chimerism after two unrelated donorUCB units were transplanted in a patientwith accelerated phase CML. Conditioningconsisted of cyclophosphamide, total bodyirradiation (TBI) and antithymocyte globulin(ATG). Units were double HLA-DRB1-mis-matched to the patient and were mismatchedto each other at a single HLA-DRB1 locus.The total nucleated cell doses per kg per unitwere 0.7 ¥ 107 and 0.9 ¥ 107. Sixty days aftertransplant, peripheral blood neutrophilchimerism had recipient cells (3%), cellsfrom UCB1 (76%) and from UCB2 (21%).Multiple cord blood transplantation mayextend the use of this source of stem cellsto the adult population, overcoming the lim-itations imposed by the low absolutenumber of progenitor cells.

At our institution, patients without higherpriority stem cell sources (HLA-identicalrelated, one antigen-mismatched related andmatched unrelated, in order of priority) areoffered participation in a clinical trial of cordblood transplantation. In this trial, if oneUCB unit containing more than 2 ¥ 107 totalnucleated cells/kg is not available, up tothree units will be combined in order to achieve that number, allowing for up to


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GVHD in recipients of class II molecular mismatched transplants (77). The optimalduration of immunosuppression withcyclosporine or tacrolimus is not known, but these agents are generally tapered anddiscontinued after 6 months in the absenceof GVHD.

Antithymocyte globulin

In vivo T-cell depletion using Campath-1 orATG is being used by several groups as aGVHD prophylaxis strategy. A matchedcontrol study reported in abstract form sug-gested that the use of polyclonal rabbit anti-serum against human T cells, administeredon days 1–5 before unmodified unrelateddonor marrow transplant for leukemia,decreased treatment-related and overall mor-tality (61). Fifty-two patients with leukemiawere compared to matched controls treatedwith a similar regimen without antithymo-cyte globulin. All patients were conditionedwith cyclophosphamide and TBI. GVHDprophylaxis consisted of cyclosporine andmethotrexate. GVHD incidence was notreported, but non-relapse mortality was lessin the study group; the 5-year cumulativeincidence of non-relapse mortality was 6%,compared to 35% in the control cohort. The overall mortality was also decreased andno significant difference between the groupsin the risk of relapse occurred (61). The role of ATG or of anti-T-cell monoclonalantibodies in this setting, however, remainscontroversial.

GVHD prophylaxis after umbilicalcord transplantation

The ideal regimen in this setting is yet to bedefined. At our institution, we use tacrolimusand mini-methotrexate, but cyclosporine and steroids are frequently used in Europe(75, 78). Methotrexate at standard doses isassociated with further delays in engraftmentand should be avoided.

T-cell depletion

Reports on the clinical use of T-cell deple-tion first appeared in the literature in theearly 1980s. Reisner et al. (79) used soybeanlectin agglutination followed by rosettingwith sheep red blood cells to deplete T lym-phocytes from the bone marrow graft inorder to prevent GVHD in a mismatchedrelated transplant. Several methods are nowavailable for T-cell depletion. A number ofmonoclonal antibody-based techniques havebeen used, such as Campath-1 (anti-CD52,with very broad specificity, depleting T aswell as other immune cells), narrow-specificity antibodies, such as antibodies tar-geting T10B9 (a/b T-cell receptor), andbroad-specificity antibodies, such as thosetargeting CD2 ± CD3 or CD5. Other tech-niques of T-cell depletion includelectins ± sheep red blood cells or mono-clonal antibodies against CD5 and CD8,counterflow elutriation (density gradientcentrifugation) and techniques developedfor CD34+ cell selection (80). T-cell deple-tion decreases GVHD incidence. However,none of these strategies has proved to besuperior to the others, and it is unclearwhether any of them improve leukemia-freesurvival or survival after unrelated donorHSCT. In general, studies of T-cell depletionhave shown that the risk of GVHD is lower,but the benefit is offset by an increased risk of graft rejection and infection due toprolonged immunodeficiency.

A retrospective analysis using IBMTR datareviewed the outcomes of 870 patients withleukemia who received T-cell-depletedtransplants from unrelated or HLA-mismatched related donors, compared to the results of 998 non-T-cell-depleted trans-plants (62). Techniques for T-cell depletionthat did not employ narrow-specificity anti-bodies produced a similar leukemia-free sur-vival and were analyzed together (‘otherT-cell-depleted’ group). Patients receiving T-cell-depleted transplants were more likely to be younger, to have advanced leukemia

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and to have greater donor–patient HLA disparity. Results for non-T-cell-depleted, T-cell-depleted with narrow-specificity anti-bodies and ‘other T-cell-depleted’ groupswere as follows: adjusted probabilities ofgraft failure were 6 ± 2%, 10 ± 4% and19 ± 5%, respectively, significantly higher forT-cell-depleted patients; the risk of severeacute GVHD was similar for the two T-cell-depleted groups and significantly lower thanwithout depletion: 35 ± 4%, 19 ± 4% and21 ± 5%, respectively. The 100-day trans-plant-related mortality was not statisticallydifferent for the non-T-cell-depleted andnarrow-specificity antibody groups, but was significantly higher for recipients of‘other T-cell-depleted’ transplantation. Com-pared with non-T-cell-depleted transplants,the adjusted 5-year probabilities ofleukemia-free survival were 31 ± 4% for recipients of non-T-cell-depletedmarrows, 29 ± 5% (P = NS) for subjectsreceiving transplants T-cell-depleted bynarrow-specificity antibodies and 16 ± 4%(P < 0.0001) after transplants depleted byother techniques. Interestingly, the risk ofchronic GVHD was higher with transplantsof T-cell-depleted marrows using narrow-specificity antibodies. The results of survivalprobabilities were similar to those ofleukemia-free survival. Therefore, despitethe reduced acute GVHD rate, no advantagein terms of leukemia-free survival or overallsurvival could be documented with T-cell-depleted BMT when compared to unmodi-fied marrow transplants using cyclosporineand methotrexate for post-BMT immuno-suppression. A multicenter, prospective ran-domized trial addressing the use of T-celldepletion with T10B9 in unrelated donorHSCT is currently in progress.

CONDITIONING REGIMENS

A wide variety of pretransplant conditioningregimens (also termed preparative regimens)

have been studied. They include combina-tions of chemotherapy agents with orwithout radiation or biologic agents. Morerecently, radioimmune conjugates and atten-uated dose regimens have been incorpo-rated in the HSCT practice with the goal ofreducing toxicity. Very few controlled trialshave been published comparing the toxicityand efficacy of different preparative regi-mens, especially in the context of unrelateddonor transplantation.

A myeloablative regimen makes HSCTmandatory in order to avoid lethal and prolonged pancytopenia. The immunosup-pressive potency of the regimen is anotherimportant consideration. Regimens employ-ing TBI, purine analogs and high-dosecyclophosphamide, with or without ATG, areprofoundly immunosuppressive and are frequently used in the unrelated marrow orcord blood transplant context. The source ofstem cells is another factor to be taken intoconsideration. Matched sibling HSCT recipi-ents, or patients undergoing autologousBMT, can tolerate more intensive condition-ing regimens (81, 82). Disease-related factorsare also important in the process of condi-tioning regimen selection. If there is clearevidence of a graft-vs.-malignancy effect,one may not need to use intensive mye-loablation and may favor immunosuppres-sion for engraftment without the need forpotentially toxic high-dose approaches. Theamount of disease to be treated may requirehigh-dose chemotherapy and/or combina-tion of agents in order to cytoreduce therecipient. Patient tolerance to the potentialtoxicities of a given regimen should be antic-ipated; for example, patients who receivedinvolved field radiation may not be able toreceive TBI-based preparative regimens andpatients with Fanconi’s anemia cannot toler-ate intensive regimens because of intrinsicdefective DNA repair mechanisms (81).

TBI-containing regimens vary in their tox-icity profiles and efficacy. Single-dose TBI ismore toxic to the lung, gastrointestinal andrenal tissue compared to fractionated dose


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TBI. Higher fractionation decreases the tox-icity and increases the maximum tolerateddose. Antileukemia activity is higher withdoses greater than 1000 cGy and engraft-ment is maximized with the addition ofcyclophosphamide. Doses of 1200 cGy or ≥ 1320 cGy, depending on the HLA matching status, with cyclophosphamide 60 mg/kg/day for 2 days, resulted in 5% ofgraft failures in the Seattle experience withpatients diagnosed with CML in the chronicphase and treated with unrelated donormarrow transplants (46). In a large series of NMDP-facilitated transplants for CML,primary and late graft failure occurred in 8%and nearly 7%, respectively (63). TBI-basedregimens had greater efficacy in promotingengraftment than busulfan-containing regi-mens. Busulfan dose adjustment may benecessary, however, to ensure adequateexposure to the drug.

Nonmyeloablative regimens

The use of decreased intensity conditioningregimens in order to explore the graft-vs.-tumor effect, minimize toxicity and extendthe use of HSCT to elderly or medicallyinfirm patients is increasing rapidly. It isbased on the use of regimens that areimmunosuppressive enough to allow donorcell engraftment, without ablation of therecipient’s hematopoietic system. Reportedtrials have involved mainly patients withsibling donors.

Giralt et al. (55) published the MD Ander-son Cancer Center experience with the useof less intensive conditioning for related andunrelated donor transplants. A population ofpatients with hematologic malignancies, otherwise not eligible for HSCT due tomedical conditions or older age, weretreated. Seventy-eight patients received fludarabine, 25 mg/m2 daily for 5 days, incombination with melphalan, 180 mg/m2 or140 mg/m2. Eight patients received cladri-bine, 12 mg/m2 for 5 days, with melphalan,

180 mg/m2. GVHD prophylaxis consisted of tacrolimus and ‘mini’-methotrexate. Fortypatients received unrelated donor trans-plants, serologically matched for HLA-A,HLA-B and molecularly matched for HLA-DRB1. The non-relapse mortality rate at2 years was 44.7%. Patients receiving unre-lated donor transplants had a trend towardsa higher risk for severe acute GVHD: 39%vs. 19% (P = 0.10). Deaths from acute GVHDoccurred more frequently in recipients ofunrelated donor transplants than in otherdonor types (11 of 40 vs. 4 of 46; P = 0.026).In univariate analysis, receiving a transplantfrom an unrelated donor was moderatelyassociated with increased mortality, com-pared to receiving a transplant from a siblingdonor, but this was not confirmed by multi-variate analysis. Therefore, the use of unre-lated donor BMT in this high-risk, olderpopulation was not an independent riskfactor for death, and engraftment wasachieved in the majority of cases. While toxicity is decreased, GVHD remains a majorproblem to be overcome. A promisingapproach has been reported in abstract formby Peggs et al. (83). They used Campath-1H,20 mg/day on days -8 to -4, with reducedconditioning with fludarabine and mel-phalan, 140 mg/m2, and cyclosporine Amonotherapy post-transplant. Only five of 30 patients developed grade I or II acuteGVHD and all 30 patients engrafted. Actuar-ial transplant-related mortality at 1 year was25% and 21 of the 30 patients are alive andprogression free with a median follow-up of5 months.

Several approaches are being pursuedworldwide. The Seattle group has describedthe use of low-dose (200 cGy) TBI as apreparative regimen for non-ablative stemcell transplantation, with a low incidence of myelosuppression. The regimen was im-munosuppressive enough to allowengraft-ment in the majority of HLA-identical siblingtransplants (84), with increased engraftmentfailure for unrelated donor cells. Slavin et al.

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(85), in Israel, pioneered the use of lessintensive conditioning regimens with flu-darabine in combination with busulfan, withpromising results in a lower risk popula-tion. Comparative studies between differentreduced intensity conditioning regimens arenot available and it is not clear if any regimenis superior or better tolerated than another(55). Therefore, non-myeloablative condi-tioning regimens allow engraftment of unre-lated donor cells and are associated with lesstoxicity and possibly with less GVHD, giventhe decreased release of cytokines and otherproinflammatory mediators. This is a rapidlyevolving area of clinical and basic researchand major breakthroughs are to be expectedin the near future.

OUTCOME OF UNRELATEDDONOR TRANSPLANTATION

Unrelated donor HSCT is generally associ-ated with an increased incidence of acuteand chronic GVHD, graft rejection andfailure and lower overall survival when com-pared with HLA-matched sibling donorHSCT. This comparison should consider thefact that only recently have unrelated bloodand marrow transplants been performed inlower risk patients. In addition, delays in the referral and in the search process haveresulted in transplants being performed inadvanced stage patients, often exposed tomultiple treatments (13, 82). The influenceof disease stage on outcome is illustrated bythe fact that, for patients treated with HLA-identical sibling transplants in 1997–1998and reported to the IBMTR, 100-day mortal-ity rates were around 10% for subjects withacute leukemia in first remission and 40% forthose with advanced disease (1).

Clinical results have to be interpreted inthe light of several variables, including con-ditioning regimen, marrow T-cell depletion,prophylaxis for GVHD and post-transplant


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immunosuppression, prophylaxis againstinfections, supportive care and the experi-ence of the transplant team. The age of therecipient and the level of histocompatibilitybetween the donor and the patient are themost significant factors influencing theresults of unrelated HSCT. Both recipient anddonor age correlate with the risk of GVHDand transplant-related mortality (46, 64, 86).Increasing donor age may be associated with an increased risk of graft failure andGVHD (58, 87). Cytomegalovirus (CMV)status, diagnosis and disease stage, timefrom diagnosis to transplant, recipient per-formance status and presence of active infec-tion at the time of transplant have beencorrelated with survival and other outcomesafter unrelated donor BMT. In the EBMTexperience, transplant center size is animportant variable, correlated with improvedoverall survival and reduced transplant-related mortality in unrelated donor trans-plants. This effect was observed only incenters transplanting more than 12 patientswith CML per year (88).

The IBMTR reported that, for all types oftransplants, the proportion of patients overthe age of 40 years is increasing. Patientsover 50 years of age account for 10% of allo-geneic transplants reported to the IBMTR (1).Establishing an upper age limit for unrelateddonor transplant is difficult. This is a rapidlyevolving field and recommendations changewith time and from institution to institution(89). Older patients should not receive mis-matched unrelated hematopoietic stem cells,however, and transplants should be per-formed in clinical trials designed to reducethe intensity of the conditioning regimen. Atour institution, the upper age limit for a fullyablative matched unrelated donor transplantis currently 50 years.

Chronic myeloid leukemia

Allogeneic HSCT is the only proven curativetreatment for CML, and this disease is the


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most frequent indication for allogeneic unre-lated BMT worldwide. Favorable prognosticfactors for survival after unrelated donorHSCT include early disease stage, CMV-seronegative recipient, HLA-DRB1 match,age less than 50 years, time from diagnosisto HSCT less than 1 year, a low body weight index and development of mild or no GVHD (46, 63). The total nucleated celldose seems to be an important predictor of survival, as it is for acute leukemiastreated with unrelated donor transplantation.A significant decrease in non-leukemiadeaths was documented recently in patients receiving a cell dose greater than 3.65 ¥ 108

cells/kg (90).The multicenter experience of 1423 trans-

plants for CML facilitated by the NMDP wasreviewed by McGlave et al. (63). They doc-umented a low incidence of hematologicrelapse of 6% at 3 years, suggesting that apotent antileukemia effect was present forpatients with early stage disease. T-celldepletion, nonetheless, was associated witha higher relapse rate among chronic phasepatients and a high relapse rate was seenamong patients in accelerated and blastphases. A subgroup of patients younger than35 years in chronic phase, transplantedwithin the first year postdiagnosis, receivingHLA-matched marrow, had a 63% disease-free survival at 3 years (95% CI, 53–73%). Asseen very often in comparisons between theresults of multicenter studies and those fromindividual institutions, single centers havereported improved outcomes that may beexplained by careful selection of transplantcandidates and possibly by increased expe-rience with unrelated donor transplantation(46, 59, 88). Table 2 summarizes the resultsof selected studies.

An NMDP sponsored study recently com-pared the outcome of 450 allogeneic trans-plants using HLA-identical sibling donors vs.2464 matched unrelated donors. Siblingdonor transplant recipients were younger(median 36 years vs. 39 years), had treatmentperformed earlier after diagnosis (7 months

vs. 17 months) and while in chronic phase(82% vs. 67% of the recipients of unrelateddonor marrow). The relative risk of severeacute GVHD was higher with unrelateddonor transplant, older donors and withtransplant performed more than 1 year fromdiagnosis. Contrary to expectations of agreater GVL effect with unrelated donorcells, the relapse rate was similar in bothgroups. A worse disease-free survival withunrelated donor BMT was documented withtransplants beyond the first chronic phaseand treatment performed more than 1 yearafter diagnosis, older recipients and donors,and CMV-seropositive recipients. However, a statistically similar 5-year disease-free sur-vival was observed for patients transplantedin early chronic phase in all age groups(younger than 30 years, 30–40 years or olderthan 40 years) (87).

The effect of prior interferon-a (IFN)therapy on the outcome of HLA-identicalsibling BMT for chronic phase CML was ana-lyzed by Giralt et al. (91) on behalf of theIBMTR. With a median duration of IFNtherapy of 2 months, a higher risk of engraft-ment failure was detected among IFN-treatedpatients when compared to hydroxyurea-only-treated subjects (2% and 0.2%, respec-tively). Patients who received IFN had alower relapse risk, but there was no differ-ence in survival, disease-free survival or non-relapse mortality. Another retrospectiveregistry study evaluated IFN in the contextof unrelated donor transplants for chronicphase CML (92). Patients who received IFNpre-BMT (n = 480) had a longer intervalfrom diagnosis to transplant than patientsnot exposed to the drug (n = 260):17 months vs. 10 months, respectively. Themedian duration of treatment with IFN was9.6 months and discontinuation occurred ata median time of 73 days pretransplant in65% of the patients from whom data wereavailable. IFN use pretransplant did notinfluence survival, leukemia-free survival,transplant-related mortality, engraftment rateor the incidence of chronic GVHD. During


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Ref. Disease No. of Donor Source of GVHD Relapse Overall aGVHDstage patients stem cells prophylaxis survival and cGVHD

(1) < 1 year 2830 HLA- BM NA NA 67% ± 2%* NAfrom identicaldiagnosis sibling> 1 year 1595 HLA- BM NA NA 57% ± 3%* NAfrom identicaldiagnosis sibling< 1 year 403 Matched BM NA NA 50% ± 5%* NAfrom unrelateddiagnosis donor> 1 year 897 Matched BM NA NA 40% ± 4%* NAfrom unrelateddiagnosis donor

(46) CP 196 MUD BM CsA/MTX 10%††† 57%** cGVHDSerologically (extensive)typed for = 67%HLA-A,-B,-DR /retrospectivetyping for-DRB1

(63) CP1 916 MUD BM CsA/other = 68% 5.7% relapsed 37.5%* aGVHDCP2 97 Serologically T-cell at 3 years gd III–IV = 33%AP/BP 402 typed for depletion = 23% cGVHDTotal† 1423 HLA-A,-B, Other = 9% (extensive)

-DR / = 60%retrospectivetyping for-DRB1 in37% ofcases

(9) CP1 1650 NA BM NA NA 39% ± 3%†† NACP2/AP 658 20% ± 4%††BP 140 5% ± 5%††

(87) CP1 369 HLA-identical 8% ± 3%sibling

1650 Matched BM 5% ± 1%unrelated P = 0.42 Severedonor RR = 1.91 aGVHD

(95% CI RR = 1.22> CP1 81 HLA-identical 18% ± 2% 1.61–2.28) (95% CI

sibling for MUD 1.05–1.59)814 Matched BM 22% ± 7% for MUD

unrelateddonor

Table 2Unrelated donor HSCT for chronic myeloid leukemia

CP: chronic phase; AP: accelerated phase; BP: blast phase; BM: bone marrow; NA: not available; HSCT: hematopoietic stem cell trans-plantation; GVHD: graft-vs.-host disease; HLA: human leukocyte antigen; RR: relative riskCsA: cyclosporine; MTX: methotrexate; aGVHD: acute; gd: grade; cGVHD: chronic; MUD: matched unrelated donor† Total of evaluable patients; 388 patients were excluded from the analysis due to incomplete data* Probability of survival at 3 years** Probability of survival at 5 years†† Probability of survival at 4 years (June, 1999)††† Probability at 5 years


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the first year post-BMT, subjects whoreceived IFN within 90 days of transplanta-tion had lower relapse rates. Patients whohad IFN stopped more than 90 days beforethe procedure had more acute GVHD andhigher relapse rates more than 1-year post-BMT than patients not treated with IFN.

Considering that the course of CML is pre-dictable in the majority of cases, with‘sudden’ onset of blast crisis being anunusual event, it has been suggested thatthere is no major adverse impact on delay-ing HSCT for 12 months in order to assesscytogenetic response to IFN or IFN-basedcombinations (93). The role of STI571 is cur-rently under active investigation.

Unrelated donor transplantation is aneffective treatment for CML, especially whenperformed in early chronic phase. Patientsand physicians, however, have to be awareof the possibility of treatment-related toxic-ity and of acute and chronic GVHD. IFN mayinduce cytogenetic remissions and prolongsurvival in CML, and the choice of the idealtreatment is very difficult, especially forrecently diagnosed patients in early chronicphase. Outcomes with transplantation areexpected to improve further during the nextdecade, reflecting advances in HLA typingand donor selection, the development of less toxic conditioning regiments and GVHDprophylaxis.

Acute myeloid leukemia

Induction chemotherapy can achieve remission in a significant majority of patientswith acute myeloid leukemia (AML). Unfor-tunately, only 20–40% will become long-term survivors (94). Consolidating remissionwith BMT may offer an increased chance ofdisease-free and survival rates. For theminority of patients with an HLA-identicalsibling, the timing of transplantation is themajor question. Patients without a matchedrelated donor may be offered autologous orunrelated donor HSCT and, occasionally, amismatched related donor (95). Other inves-

tigational options include haploidentical andcord blood transplants.

Older age is a major negative prognosticfactor for complete and durable remissionsin AML, and the peak age incidence of thisdisease coincides with the traditional ageboundaries for the performance of HSCT.Therefore, part of the results attributed toHSCT, especially in first remission, may berelated to selection bias.

Given the high remission rate and pro-longed remissions achieved with chemother-apy, there is no clear role for HSCT forpatients with t(8;21) and inversion 16. Alltrans retinoic acid (ATRA) and chemother-apy produce high remission and cure ratesfor patients with promyelocytic leukemia,without the need for BMT. High-risk featuresat presentation may indicate the use of allo-geneic or autologous BMT in remission, suchas high white cell count, more than onecourse of effective induction therapy toachieve a response and chromosomal abnor-malities such as deletion(5) or deletion(7).The presence of persistent minimal residualdisease is another tool that can be used toselect patients who may benefit from earlytransplantation, with a low burden ofleukemia, but this is an evolving area andno conclusions can be drawn at this point.Cassileth et al. (96, 97) did not find anadvantage in disease-free survival or overallsurvival with the use of either autologous or allogeneic BMT, when compared to asingle course of high-dose cytarabine (Ara-C), but other studies suggested betteroutcomes with BMT.

Patients in early relapse should probablyreceive HSCT without salvage chemo-therapy. For subjects with high marrow andperipheral blood blast counts, achievementof a second remission with chemotherapyprior to BMT may improve outcomes. These are areas of considerable debate,however.

The benefit of an effective GVL effect after unrelated donor allografts is frequentlyoffset by higher transplant-related mortality.


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Pooled registry data show that results withautologous BMT for AML, with lower trans-plant-related mortality and higher recurrencerates, are usually superimposable on thoseof unrelated donor transplantation. A singleinstitution study with acute leukemiapatients showed 40% of deaths occurring in remission after unrelated HSCT vs. 28%after autologous BMT (98). The positiveeffect of a younger age and a higher doseof marrow cells in patients treated with unre-lated donor HSCT has been documented(64, 99–101).

A recent abstract compared the results of684 autologous transplants reported to theAutologous Bone Marrow Transplant Reg-istry (ABMTR) with 515 unrelated donortransplants reported to the IBMTR and theNMDP for AML in first or second remission(102). This study is very illustrative of the dif-ficulties in comparing groups of patients thatprobably differ in several other ways thatcannot be assessed by the registries. Themajority of autologous transplants (73%)were performed in first remission, asopposed to 44% of unrelated transplants.The latter group contained patients withpoorer performance status and higher riskdiseases. After a median follow-up ofapproximately 4 years, the 3-year cumulativeincidences of transplant-related mortalityafter BMT in first or second remission weresignificantly higher after unrelated donortransplants, while the risk of relapse was sig-nificantly higher after autologous BMT (102).

Unrelated donor transplantation for AMLis effective and associated with a strong GVLeffect. While it certainly has to be consid-ered for patients beyond first remissionlacking an HLA-identical sibling, its role forpatients in first remission is not defined. It isappropriate to discuss autologous marrowharvesting in first remission, without evi-dence of minimal residual disease. The deci-sion to pursue autologous BMT vs. unrelateddonor HSCT is often based on local experi-ence and the physician’s perception of thedisease-related risk, and firm guidelines are

not yet available. Table 3 reviews the resultsof studies of unrelated donor transplants for AML.

Acute lymphoid leukemia

Age has a great influence on the duration ofremission and overall survival for ALL, withadults having a poorer life expectancy thanchildren. The prevalence of the Philadelphiachromosome and other poor prognostictranslocations increases with age, reflectingdifferences in disease biology. The proba-bilities of 5-year survival and 5-year remis-sion rates were 39% and 38%, respectively,in a group of adult patients (median age,39.5 years) treated with the hyper-CVADregimen (103). The impact of different prog-nostic factors for the achievement of remis-sion, however, may differ according to theintensity of the induction regimen utilized(104, 105).

Patients with recurrent or high-risk ALLcan be cured by HSCT but, as with AML,questions remain in most cases regarding the timing of transplantation. IBMTR datashow that the most cited indications for BMTin first remission are older age, high white cell count at diagnosis, chromosomal abnor-malities, such as the Philadelphia chromo-some or t(4;11), and delayed achievement of first remission (1).

Unrelated donor transplant recipients hada 3-year probability of survival of 44 ± 7%for transplants performed in first remissionreported to the IBMTR (Table 4). Outcomesof autologous transplants in first remissionwere similar. Again, the limitations of reg-istry data have to be taken into considera-tion when analyzing these results. Thecriteria used by different centers whendeciding the type of transplant to beemployed vary widely, in ways that cannotbe accounted for in registry data analysis.Weisdorf et al. (106, 107) compared resultsof autologous and unrelated donor trans-plants using registry data. Unrelated donortransplant recipients had more higher risk


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karyotypes (21% vs. 8%) and more patientswith white cell counts greater than50 ¥ 109/L (35% vs. 14%) than autologoustransplant patients. A lower relapse rateoccurring after unrelated donor BMT wasoffset by a significantly higher transplant-related mortality, and outcomes at 3 yearswere similar for the two groups.

The Seattle group reported their experi-ence treating 18 Philadelphia-positivepatients with unrelated donor HSCT (57).The group included pediatric patients, withan overall median age of 25 years (range,1.7–51 years). Of the seven patients in firstremission, six were alive, without disease, at the time of publication. All patients were conditioned with cyclophosphamideand TBI, with GVHD prophylaxis usingmethotrexate with either cyclosporine ortacrolimus. Grade III–IV acute GVHDappeared in six of 17 evaluable patients andchronic extensive GVHD in seven of 13patients at risk. The likelihood of leukemia-free survival at 2 years was 49 ± 12%. Thirty-

eight per cent of 33 Philadelphia-positivepatients treated with HLA-identical siblingtransplants, beyond first remission, achievedsustained disease-free survival in the IBMTRexperience (108). Allogeneic transplantsshould be performed in first remission forpatients with Philadelphia chromosome-positive ALL, but other indications of BMTin first remission are not clearly defined.

The results of unrelated donor BMT forALL patients not in remission are usuallyvery poor and attempts should be made totransplant after salvage chemotherapy. In the NMDP experience, the 4-year survival ofpatients not in remission is only 6 ± 3% (9).

Myelodysplastic syndrome

As with other indications for unrelated donortransplantation, the use of this form of treat-ment for myelodysplastic syndrome (MDS) issurrounded by controversy and well-definedguidelines regarding the timing of transplan-tation are not available. The overall survival

Reference Disease stage Number Donor/source TRM Relapse Overallof patients of stem cells survival*

(1) 1st CR 3424 HLA-identical 60% ± 2%≥ 2nd CR 883 sibling BMT 40% ± 4%1st CR 649 (patient total) Unrelated donor 32% ± 6%≥ 2nd CR BMT 43% ± 7%1st CR 1223 Autologous 55% ± 3%≥ 2nd CR 513 35% ± 5%Not in CR 275 18% ± 5%

(102) 1st CR 499 Autologous 8% (6–11) 38% (34–43)227 Unrelated donor 44% (38–51)† 14% (10–19)†

BMT2nd CR 185 Autologous 14% (9–20) 45% (37–53)

288 Unrelated donor 53% (46–59)† 13% (10–18)†BMT

(9) 1st CR 264 Unrelated donor 25% ± 7%**2nd CR 344 BMT 28% ± 6%**≥ 3rd CR 741 9% ± 3%**+ no CR

Table 3Unrelated donor transplantation for acute myeloid leukemia

TRM: transplant-related mortality; CR: complete remission; NA: not available.* Probability of survival at 3 years.** Probability of survival at 4 years (June, 1999).† P < 0.001 for the comparison of autologous vs. unrelated donor BMT.


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of 272 patients with refractory anemia orrefractory anemia with ringed sideroblastsundergoing matched sibling allogeneic BMTwas 53 ± 7% at 3 years in the IBMTR experi-ence (1). The likelihood of survival forpatients with refractory anemia with excessblasts (RAEB), refractory anemia with excessblasts in transformation or chronic myelo-monocytic leukemia was 36 ± 4%. The NMDPreports a 4-year survival probability of26 ± 4% for 578 recipients of unrelated donorHSCT, while IBMTR data show a 3-yearoverall survival of 23 ± 6% among 314patients receiving unrelated transplants (9).Castro-Malaspina et al. (109) reported 320patients treated with allogeneic unrelateddonor transplants. The median age was37 years, markedly less than the peak ageincidence of MDS. The disease-free survivalat 2 years was 30%, with 24% probability ofrelapse. A worse prognosis for patients with

MDS ‘in transformation’ has been docu-mented in a single institution study (110). Nodifference in survival or in risk of relapse wasobserved in another series with the use of T-cell depletion for GVHD prophylaxis (111).

A major goal of research is to extend theuse of allogeneic transplants to the majorityof patients with MDS, namely those olderthan 50 years. The use of non-myeloablativeconditioning regimens is being activelyinvestigated by our group and other institu-tions in this setting.

Aplastic anemia

Allogeneic HSCT is the treatment of choicefor patients with severe aplastic anemia whohave an HLA-identical sibling. Unfortunately,it has been estimated that only 20–30% ofpatients have a fully HLA-matched siblingdonor. The IBMTR data on 1754 HLA-

Reference Disease stage Number Donor/source TRM Relapse Overallof patients of stem cells survival*

(1) 1st CR 1549 HLA-identical 52% ± 3%≥ 2nd CR 1511 sibling BMT 42% ± 3%1st CR 337 Unrelated donor 44% ± 7%≥ 2nd CR 844 BMT 33% ± 3%1st CR 185 Autologous 43% ± 9%≥ 2nd CR 268 37% ± 7%Not in CR 50 15% ± 11%

(9) 1st CR 282 Unrelated donor 34% ± 8%**2nd CR 487 BMT 32% ± 5%**≥ 3rd CR 232 24% ± 6%**No CR 341 6% ± 3%**

(106)† 1st CR Autologous 20% ± 12% 49% ± 12% 44% ± 12%Unrelated donor 47% ± 7% 15% ± 4%†† 47% ± 6%§BMT

2nd CR Autologous 9% ± 5% 64% ± 8% 32% ± 9%Unrelated donor 46% ± 5% 23% ± 4%†† 36% ± 5%§BMT

Table 4HSCT for acute lymphoid leukemia

TRM: transplant-related mortality; CR: complete remission; NA: not available; HSCT: hematopoietic stem cell transplantation.* Probability of survival at 3 years.** Probability of survival at 4 years (as of June, 1999).† Age younger than or less than 50 years; unrelated donor patients were significantly younger (median 13 years vs. 18 years, P < 0.0001).†† P < 0.001.§ P = NS.

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impacted on survival (114). In order to usemismatched related or unrelated donors,conditioning regimens that are more inten-sive than those used in the HLA-identicalsibling setting appear to be indicated(114, 115). Graft failure is a potentialproblem and has been reported in 0–50% of studies (112).

Patients with severe aplastic anemia wholack a matched related donor may benefitfrom unrelated donor HSCT. Age, time inter-val from diagnosis to transplant and extentof donor–recipient HLA matching are themost important variables to be considered.The role of cord blood transplantation andother alternative donor transplants in thiscontext is yet to be explored.

Other diseases

Unrelated donor transplantation has beenused for the treatment of other diseases, withvariable success. Pediatric patients maybenefit from this form of treatment forFanconi’s anemia or inherited disorders ofmetabolism (1, 9). As of June, 1999, 201patients had received unrelated donor trans-plants for non-Hodgkin’s lymphoma, with a 4-year probability of survival of 24 ± 7%(9). We and other groups are investigatingthe use of unrelated HSCT to treat patientswith chronic lymphocytic leukemia andHodgkin’s disease.

IMMUNE RECONSTITUTIONAND INFECTIOUS

PROPHYLAXIS

The post-transplantation period is character-ized by several immune system deficiencies.B- and T-lymphocyte functions are affectedand the frequent development of acute andchronic GVHD adds other disruptions ofhost immunity. Inversion of the normalCD4/CD8 T-cell ratios is common andusually persists for 1 year, as subpopulations


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identical sibling transplants performed be-tween 1991 and 1997 showed a 3-year prob-ability of survival of 74 ± 3% for 978 patientsyounger than 20 years and 65 ± 3% for 776patients older than 20 years. Patients surviv-ing more than 2 years post-BMT have anexcellent 7-year probability of survival of94 ± 1% (1).

Registry data on unrelated donor trans-plantation for aplastic anemia show poorerresults than those reported after HLA-identical sibling donor transplantation. Unre-lated donor HSCT is usually reserved forsubjects failing one or more courses of inten-sive immunosuppression with ATG and/orcyclosporine or for patients developing MDS(112). The 3-year likelihood of survival forrecipients of unrelated marrow was 46 ± 7%in 239 patients younger than 20 years and36 ± 12% in 71 older patients reported to theIBMTR (1). The 4-year probability of survivalfor 288 patients whose transplants were facilitated by the NMDP was 36 ± 7% (9).Results of unrelated donor transplantationfor aplastic anemia appear to be significantlybetter among pediatric patients (113).

Deeg et al. (114) reviewed the NMDPexperience in 141 patients with severe aplas-tic anemia who received transplants between1988 and 1995. Eighty-six per cent ofpatients received TBI-based conditioningregimens and 14% were treated with chemo-therapy-based regimens. GVHD prophylaxisconsisted of a cyclosporine-containing com-bination in all but 13% of patients, while 32% received T-cell-depleted marrow. Recip-ients of a serologically matched marrow hadbetter outcomes than patients given a mis-matched transplant. The survival of recipi-ents of marrows serologically matched atHLA-A and HLA-B, and molecularly matchedfor HLA-DRB1, was significantly better thansurvival of HLA-DRB1-mismatched patients(56% vs. 15% surviving at 3 years). GradeII–IV acute GVHD developed in 52% ofpatients and extensive chronic GVHD in 24patients at risk (31%). Delay of transplant formore than 3 years from diagnosis negatively


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of lymphocytes recover. Secretory imm-unoglobulin A (IgA) production may bedecreased for many years after unrelateddonor BMT. Treatment for chronic GVHDdelays the recovery of a normal CD4/CD8ratio and of responses to neoantigens, andprolongs abnormal immunoglobulin profilesand hypogammaglobulinemia (116).

This series of immune deficiencies usuallytranslates into increased risk of infections byencapsulated bacteria and viruses, e.g. her-pesviruses. The majority of cases of varicellazoster (VZV) occur during the first year post-BMT, but patients in chronic immunosup-pression are at greater risk for prolongedperiods of time. Adenovirus-induced cystitisor pneumonia is a feared complication thatusually occurs earlier after transplant.Poliomavirus may also cause cystitis. Epstein–Barr virus (EBV) lymphoproliferative diseaseof donor origin is more common during the first 6 months post-BMT. A late onset form is usually of recipient origin (117).Pneumocystis carinii infection may occur atany time in patients on chronic immuno-suppression. Prophylactic trimethoprim–sulfamethoxazole is very effective in preventing pneumonia by P. carinii and acyclovir is usually effective in minimizingand preventing VZV and herpes simplex virusreactivations. Respiratory syncytial virus(RSV) pneumonia is associated with a mortal-ity rate of 30–70% among transplant re-cipients (118). Treatment with aerosolizedribavirin may be beneficial.

Infections with CMV occurred in up to30% of patients before the availability ofCMV prophylaxis. Pancytopenia, retinitis,pneumonia, colitis, hepatitis and encephali-tis have been associated with CMV. CMVserologic status is often a donor selection criterion, especially if the recipient is CMV-negative. CMV-negative blood donors andthe use of leukodepletion filters are equallyeffective in reducing the transmission ofCMV through blood products. Ganciclovirprophylaxis is frequently used, but its impacton survival remains to be demonstrated

(119). Ganciclovir is associated with pancy-topenia and delayed immune reconstitutionto CMV. Pre-emptive therapy with ganci-clovir should be based on CMV antigenemiaor possibly PCR methods (120); it has beenshown to decrease the incidence of CMV-induced pneumonia (117). Specific cytotoxicT cells have been generated ex vivo andsafely infused in patients. Their efficacy inpreventing CMV disease is currently beingassessed (121).

At our institution, we perform a CMV anti-genemia test twice per week until day 100.Antigenemia is considered to be positive ifmore than (or equal to) three positive cellsare found in low-risk recipients (recipient ofmatched sibling transplant, no GVHD) andif more than (or equal to) one cell is detectedin high-risk recipients (all others). If whiteblood cells are < 1.0 ¥ 109/L, then any pos-itive cells is considered to be a positive reac-tion. CMV cultures from urine, throat andsputum are not performed for monitoring,but are ordered as clinically indicated. Allallogeneic recipients who are CMV-sero-positive or who have a CMV-seropositivedonor receive ganciclovir pretransplant.CMV-seronegative recipients with seronega-tive donors do not receive prophylactic ganciclovir pre- or post-BMT, but receiveacyclovir. Ganciclovir prophylaxis is admin-istered if the donor or recipient is CMV-positive and is receiving a haploidenticaltransplant or if the patient develops GVHDrequiring steroid treatment. Prophylaxisbegins postengraftment. CMV-seropositivepatients receiving unrelated marrow or cordblood transplants are given post-transplantacyclovir and are started on therapy if CMVantigenemia occurs. If they develop GVHDrequiring steroid or ATG treatment, ganci-clovir prophylaxis is given. Unrelated donorand mismatched transplants should receiveintravenous immunoglobulin prophylaxis.For patients with acute GVHD receivingsteroids or other immunosuppressive treat-ment, ganciclovir prophylaxis and intra-venous immunoglobulins are given weekly

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until day 90. In case of intolerance to gan-ciclovir, foscarnet should be used.

Our current practice is to give patientsprophylactic fluoroquinolone and oral penicillin starting pretransplant. Patients are treated preferably in laminar airflow ventilation rooms and receive prophylacticfluconazole. However, infections caused bythe invasive mold Aspergillus sp. remain aproblem despite the use of prophylactic fluconazole.

Prophylactic trimethoprim–sulfamethoxa-zole is given pre-BMT until day – 2 and isrestarted after stable engraftment occurs.Patients receive prophylaxis with flucona-zole, trimethoprim–sulfamethoxazole andvalacyclovir while on immunosuppressivemedications.

GRAFT FAILURE

Sustained engraftment of donor hematopoi-etic cells is dependent on several factors.Genetic disparity is a major determinant ofgraft failure and rejection, and was analyzedin a previous section. Patient diagnosis,allosensitization, intensity of the pretrans-plant conditioning regimen and post-transplant immunosuppression therapy arefactors known to be involved. The dose ofhematopoietic stem cells is very importantfor successful engraftment and lower celldoses increase the likelihood of graft rejec-tion. A relative risk of 4.9 for each decreaseof 1 ¥ 108 nucleated cells/kg has been sug-gested (46). Other causes of graft failureinclude T-cell depletion, infections and med-ications. In a large cohort of CML patients,multivariate analysis showed a lower risk ofprimary graft failure with the use of TBI-containing preparative regimens, a nucleatedcell dose of more than 2.1 ¥ 108 cells/kg,transplant in chronic phase and an HLA-matched donor (63). The use of preparativeregimens without TBI may be associatedwith a higher rejection rate.


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Graft rejection after HLA-identical HSCTcan be caused by the recipient’s immuno-competent T lymphocytes recognizing minorhistocompatibility antigens on donor stemcells, determined by genes outside the MHC.During rejection of a male donor graft by afemale recipient, two male (H-Y)-specificcytotoxic T-lymphocyte clones were identi-fied in the recipient’s blood (68). CytotoxicT lymphocytes against several H-Y antigenscan contribute simultaneously to graft rejec-tion after HLA-identical, sex-mismatchedHSCT and are likely to be involved in similarprocesses during matched unrelated donortransplants. Host bone marrow stromal cellsprovide a matrix for donor hematopoieticcells, but donor alloreactive cytotoxic T lymphocytes may lyze these cells, damagingthe marrow microenvironment. This poorlyunderstood relationship might be disrupted,resulting in graft failure, as it may occurduring severe GVHD. Prior chemotherapyand radiation therapy may be involved infailure of engraftment, by means of marrowstromal damage.

Failure of engraftment has been observedafter moderately ablative conditioning andmatched unrelated donor transplants forleukemias. In this setting, the use of flu-darabine-based regimens seems to improvethe chances of donor cell engraftment(122, 123). Using a non-myeloablativeregimen consisting of 2 Gy TBI and post-BMT immunosuppression with cyclosporineand mycophenolate mofetil, graft rejectionsoccurred in 20% of patients receiving HLA-identical sibling HSCT. The addition offludarabine 30 mg/m2/day on days -5, -4and -3 eliminated graft rejection among 28 patients so treated, as opposed to 10 engraftment failures among 50 patients

Unrelated donor hematopoietic stem cell trans-plants are generally associated with increasedincidence of acute and chronic GVHD, graftrejection and failure, and lower overall survivalwhen compared with HLA matched siblingdonor stem cell transplants.

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treated without fludarabine. Four rejectionsoccurred among 33 recipients (12%) of unrelated donor transplants with fludarabineand irradiation (123).

Late graft failure has been reported in2–15% of unrelated donor transplant recipi-ents (46, 47, 63). Causes are very similar tothose involved in primary graft failure andinclude HLA incompatibility, GVHD, graft T-cell depletion, low numbers of progenitorcells, infections with CMV, EBV, herpessimplex virus, hepatitis viruses, parvovirusB19 and human herpesvirus 6 (HHV 6), aswell as a damaged bone marrow stroma.Chronic GVHD may manifest itself by per-sistent pancytopenia and graft failure, fre-quently made worse by opportunisticinfections such as CMV.

The prognosis after graft failure is usuallypoor. A second transplant may be contem-plated if the general condition of the patientis good and the donor is available. Supportwith growth factors may minimize neu-tropenia and anemia. The outcome ofsecond transplantation for primary graftfailure is probably worse than for secondarygraft failure (124).

UNRELATED DONORLYMPHOCYTE INFUSIONS

Donor lymphocyte infusions (DLI) caninduce stable remissions in patients withrelapsing leukemia after HLA-identicalsibling donor transplants. Up to 80% ofpatients with CML in cytogenetic or hema-tologic relapse in chronic phase mayrespond. The induction of remissions in this setting provides powerful in vivo proofof the existence of a graft-vs.-tumor effect.Responses to DLI have been documented in20–45% of patients with relapsing AML andin 10–15% of subjects with relapsed ALL (54).Other diseases appear to be responsive, suchas multiple myeloma, chronic lymphocyticleukemia and low grade lymphomas. Treat-


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ment-related mortality may be as high as30% and is usually related to GVHD, pancy-topenia and immunosuppression-relatedcomplications.

Better matching criteria using molecular typing willdecrease the incidence of acute and chronicGVHD, but will also reduce the likelihood of iden-tification of donors among minorities.

The experience with unrelated DLI islimited, but available data suggest similarefficacy and toxicity when compared torelated donor lymphocytes (125). A retro-spective analysis of unrelated DLI facilitatedby the NMDP in 19 centers has been pub-lished recently (126). Fifty-eight patientswith disease relapsing after unrelated donorHSCT were treated with DLI; eight patientswere treated in remission and 50 subjectswere studied for response. The cell dose wasgreater than 1 ¥ 108 mononuclear cells/kg in 48% of cases. A complete remission wasachieved in 42% of cases (95% CI, 28–56%).Complete remission rates by diagnosis wereas follows: CML in ‘early phase’, 58% (sevenof 12 patients); AML, 42% (eight of 19patients; 95% CI, 20–64%). Grade II–IV acuteGVHD developed in 25% of recipients; 41%developed chronic GVHD. Bone marrowaplasia developed in four of 34 evaluablepatients (12%). In a multivariate analysisincorporating demographic factors, HLAmatching, time from transplant to relapseand from transplant to DLI, GVHD afterBMT, graft T-cell depletion and DLI for CMLvs. other diagnosis, improved survival was associated with a time interval fromBMT to disease relapse greater than 1 year.Improved disease-free survival was associ-ated with an interval from transplant to DLIof more than 1 year. Taking into account thesmall number of patients and the differenttreatment protocols involved in 19 institu-tions, these results are not strikingly differ-ent from those observed after related donorDLI (54, 125).

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� of the problem is a lack of information givenpre-BMT, but several adaptive mechanismsare involved as well. This includes the viewof cancer as a completely reversible process,denial and unrealistic optimism. An adjustedconcept of ‘normality’ needs to be built by the BMT team and the patients in orderto minimize improbable expectations post-HSCT; this should start pre-BMT and be reinforced regularly during the recoverypost-transplant (128).

FUTURE DIRECTIONS

Unrelated donor hematopoietic stem cellscan potentially extend the benefit of trans-plantation to all patients in need. However,major obstacles remain to be overcome.Better matching criteria using moleculartyping will decrease the incidence of acuteand chronic GVHD, but will also reduce thelikelihood of identification of donors amongminorities. New methods of GVHD preven-tion and treatment are urgently needed; iftheir efficacy improves, it may be possibleto employ mismatched donors, increasingthe possibilities of performing transplants forsubjects with ‘rare’ HLA types. Beatty et al.(129) have estimated that, if a four of sixantigen HLA-A, HLA-B, HLA-DRB1 matchbecomes suitable, it will be possible to identify a donor for recipients of any racialbackground in registries with fewer than10 000 potential donors. The identification of ‘permissive antigens’ may increase flexi-bility in donor–recipient pair matching. This may be achieved through integration of international efforts and the creation oflarger HLA databases.


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PSYCHOLOGIC/QUALITY OF LIFE ISSUES

Quality of life issues after unrelated donorHSCT have been addressed by few studies.Several factors may influence the quality oflife of subjects surviving BMT, including thepresence of chronic GVHD, primary diseaseremission status, presence of social andfamily support, sexual function, employmentand ability to work. An analysis from the UKshowed that 60% of subjects surviving morethan 1 year after transplant returned to fulltime work and 15% to a part time job (65).It also depicted the negative impact ofGVHD on daily activities, strength andoverall quality of life. Of 196 patients treatedin Seattle for CML in chronic phase withunrelated donor BMT, 57% were alive at5 years (46). One year after BMT, 36% of 119evaluable patients had a Karnofsky status of100 and 86% had a score greater than 80.Three years after the transplant, 50% of 46evaluable patients had a score of 100 and98% had a score greater than 80. However,two-thirds of evaluable patients had beentreated for or had chronic GVHD. The paperdid not address the quality of life directly,but it is to be expected that chronic GVHDinfluenced the quality of life in severalaspects (46, 65).

A trend towards a worse performancestatus in patients treated with unrelatedHSCT when compared to recipients ofsibling donor BMT has been reported (48).A significant fraction of the long-term sur-vivors had chronic GVHD and were unableto work due to related symptoms. Dissatis-faction with overall appearance, physicalstrength and sexual function also correlatedwith the incidence of GVHD. The onset ofsexual dysfunction, however, usually pre-cedes the transplant, but may not be ame-liorated by HSCT (127).

Studies have identified overly optimisticexpectations for post-BMT physical perfor-mance status as a major source of stress andpoor acceptance of physical limitations. Part

New methods of GVHD prevention and treatmentare needed; if their efficacy improves, one mayemploy mismatched donors, increasing the possi-bilities of performing transplants for subjects with‘rare’ HLA types.


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�

At the MD Anderson Cancer Center, we areinvestigating whether the addition of pento-statin, a potent T-cell cytotoxic agent, totacrolimus and ‘mini’-methotrexate for GVHD prophylaxis will improve currentresults of mismatched and matched unrelatedand mismatched related donor transplants.Our group is also studying the use of multiple cord blood unit transplantation, asdiscussed in a previous section. The use ofmismatched UCB seems to be associated with less GVHD than with mismatched bone marrow or PBSC transplants. If the cellnumber ‘barrier’ is overcome, transplants may be offered to more patients in need.

Practically all patients have a haplo-identical family donor. Nevertheless, the role of this form of transplantation is yet to be defined, but it also offers hope of ex-panding the number of subjects benefitingfrom HSCT. How to integrate and prioritizethe different options of alternative donorHSCT for patients without family or matchedunrelated marrow donors is largely unknown.

New conditioning regimens are offering

the prospect of HSCT to older patients, andbasic and clinical studies on immunologictolerance will most certainly advance in thenext decade. The integration of non-transplant modalities of treatment, betterHLA typing and the use of ‘new’ sources of stem cells, with improved GVHD prophy-laxis and supportive care, is changing thescenario of HSCT. We will witness majoradvances in the coming years, as a reflectionof the efforts and investments made duringthe last 40 years in the field.

Marcos de Lima, Richard ChamplinDepartment of Blood and MarrowTransplantation, University of Texas,MD Anderson Cancer Center, Houston,Texas, USA

Correspondence: Marcos de Lima, De-partment of Blood and Marrow Trans-plantation, University of Texas, MDAnderson Cancer Center, 1515 HolcombeBlvd, Box 423, Houston 77030-4009,Texas, USA

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